Strong evidence of an etiologic association between HPV and a distinct subset of oropharyngeal squamous cell carcinomas, distinguished by the presence of high-risk HPV DNA in tumor cell nuclei, has recently been reported. Because viral oncoproteins E6 and E7 are consistently expressed in HPV-associated cancers and are tumor-specific antigens, patients with HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC) may benefit from therapeutic strategies designed to augment the cellular immune response to HPV oncoproteins. In preclinical model systems, linkage of Mycobacterium tuberculosis heat shock protein 70 (HSP70) to the HPV 16 E7 antigen was demonstrated to significantly enhanced the potency of a naked DNA vaccine. Vaccination of mice with E7/HSP70 DNA increased the frequency of HPV 16 E7-specific CD8+ T cells by at least 30-fold compared to vaccination with wild-type E7 DNA and generated a significant antitumor effect against an E7-expressing tumor. This vaccine demonstrated significant potency against established E7-expressing murine tumors with down-regulation of Major Histocompatibility Complex (MHC) class I molecules, an important finding given a significant proportion of advanced stage HNSCC exhibit down-regulation of MHC class I molecules. Furthermore, repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes (CTL) with higher avidity and improved protective anti-tumor effects. An E7 gene with mutations in the critical Rb binding residues (termed E7 detox) was generated which failed to bind Rb, had no transforming activity, and yet maintained potent immunogenicity. Support from the NIH Rapid Access to Intervention Development program has been obtained to generate clinical grade E7(detox)/HSP70 in the pNGVL4a DNA backbone for clinical trials. Sequential phase I and II clinical trials of repeated intramuscular injections of the E7(detox)HSP70 DNA vaccine are proposed in patients with HPV 16-positive oropharyngeal squamous cell carcinoma. The phase I trial is designed to evaluate the feasibility and safety of adjuvant therapy with escalating doses of E7(detox)HSP70 DNA vaccination as well as the effect of dose on the generation of HPV 16 E7- specific CD8+ T-cells in peripheral blood. The phase II trial is designed to evaluate whether neoadjuvant therapy with repeated DNA vaccination alters the number, avidity and E7-specific cytotoxic activity of HPV 16 E7-specific CD8+ T-cells and whether successful generation of these cells results in infiltration of tumor by CTL and other immune effectors. In an exploratory analysis, subjects in the phase II trial will also be observed for evidence of clinical response to E7(detox)/HSP70 DNA by measuring the size of target lesions before and after vaccination and reporting time to disease progression and two-year progression free survival.